CLONING, SEQUENCING, AND OVEREXPRESSION OF THE ANAEROBIOSPIRILLUM-SUCCINICIPRODUCENS PHOSPHOENOLPYRUVATE CARBOXYKINASE (PCKA) GENE

The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the ana erobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum s ucciniciproducens was cloned, sequenced, and expressed in Escherichia coil. The gene encoded a 532-residue polypeptide with a calculated mol ecular mass of 58.7 kDa, The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 6 7.3% identical and 79.2% similar to the E. coli enzyme, The A. succini ciproducens pckA transcription start site was determined, and putative promoter regions were identified, The recombinant enzyme was overexpr essed in E. coli, The purified enzyme was indiscernible from the nativ e enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.